PROJECT SUMMARY In the U.S., enteric infections and inflammatory bowel diseases (IBD) cause great morbidity and consume a large portion of health-care resources. Limited knowledge of the host-microbe dynamic in intestinal health and disease is a barrier for therapies aimed at boosting immunity and reducing inflammation. There is a large un- met need for fundamental understanding of the role of the intestinal epithelium in communication with gut bac- teria. The long-term goal of this project is to uncover regulatory pathways that are engaged in vivo for the in- duction of host defense genes in the intestinal epithelium. The overall objective of the present proposal is to identify upstream mechanisms that regulate the PLC-PKD-TFEB (PLC-1/DKF-1/HLH-30 in C. elegans) path- way that we identified in the previous funding period, and downstream mechanisms by which it drives gene induction. The central hypothesis is that the cholinergic nervous system controls the PLC-1/DKF-1/HLH-30 pathway, which induces a transcription factor cascade that is important for host defense in the intestine. The rationale for the proposed research is that complete understanding of how the nervous system interacts with the entire PLC-1/DKF-1/HLH-30 pathway is essential for fundamental understanding of host defense, and that a better grasp of its workings is necessary to harness its potential as a therapeutic target for inflammation or infection. To test the central hypothesis, we propose the following specific aims: 1) Determine the role of the nervous system in the activation of HLH-30/TFEB, by defining neurons that detect infection, the target distal tissues, and pathogen genes necessary for detection; 2) Define the link between DKF-1/PKD and HLH- 30/TFEB, by reverse genetics, HLH-30/TFEB phosphoanalysis, and defining protein-protein interactions; and 3) Determine the role of downstream transcription factors in mediating HLH-30/TFEB function, by intestine- specific loss of function and transcriptional profiling. The expected contribution of the proposed research is to define the role of the cholinergic nervous system in controlling the PLC-PKD-TFEB pathway and its down- stream transcription factor network during infection in vivo. This contribution is significant because it will repre- sent a leap forward for understanding the molecular basis of regulation of the epithelial barrier by the nervous system, and will enable the future development of therapies that manipulate the emerging muscarinic- controlled transcriptional network. The proposed research is extremely innovative because the PLC-PKD- TFEB pathway has not been studied in any context, and thus the proposed research is extremely innovative. The research proposed here represents a new and substantive departure from the status quo, by shifting focus to a new nervous system-intestinal signaling axis that is mediated by the newly uncovered PLC-PKD-TFEB pathway. The proposed work overcomes many barriers to the comprehensive understanding of host defense, and has great potential to accelerate the development of innovative therapies that target the emerging musca- rinic-controlled transcriptional network to treat infectious or inflammatory diseases.